The present invention relates to the preparation and use for imaging of radionuclide-labeled cells. In the last ten years, the use of blood cells labeled with radioactive tracers for diagnostic imaging has increased. The references cited in this application are incorporated by reference herein in their entirety related to the context of their citations. Labeled cells are currently being used for an ever increasing variety of applications. Erythrocytes, which are the most frequently used labeled cells, have been successfully labeled with the radioisotope .sup.99m technetium (.sup.99m Tc). .sup.99m Tc-labeled erythrocytes have been used to study cardiac function (MUGA scans) and for the detection and localization of gastrointestinal bleeding. Unfortunately, a successful procedure for labeling other blood cells, e.g., leukocytes and platelets, with .sup.99m Tc has yet to be established.
Leukocytes and platelets have, however, been successfully labeled with the radioisotope, .sup.111 indium (.sup.111 In) The use of .sup.111 In-labeled leukocytes has gradually increased since their introduction in 1976, and research in this area continues to be very active (Milgran, et al., Clin. Nucl. Med., Vol. 10, pp. 30-34, 1985). In the Milgran article, .sup.111 In-labeled lymphocytes were administered to patients with chronic inflammatory disease. Whole body gamma-ray camera scans were performed in order to image localization of the .sup.111 In-label. Localization of the .sup.111 In-label was normally imaged in the spleen, the liver, bone marrow, and the cervical and inguinal lymph nodes. Localization of the .sup.111 In-label outside of these areas was considered abnormal or positive. Patients with chronic osteomyelitis, chronic arthritic disease, or chronic bladder inflammation had positive scans.
.sup.111 In-labeled eosinophils were also used for the detection and localization of inflammatory lesions and parasitic infections which could not be detected by other diagnostic modalities. (Runge, et al., Nucl. Med. Biol. Vol. 12, No. 2, pp. 135-144, 1985). In The Runge Article, .sup.111 In-labeled eosinophils were used to image the chemotactic response of eosinophils to intradermal injections of soluble schistoscoma antigen, S. mansoni eggs, E.coli, and turpentine. Gamma-ray cameras were used to image the localization of the radiolabel. Soluble schistosoma antigen and s. mansoni eggs provided a greater stimulus for localization than E. coli or turpentine. The article suggested that .sup.111 In-labeled-eosinophil scans were more sensitive to parasitic infections than bacterial infections.
.sup.111 In-labeled leukocytes have been used in the early detection of occult infection. (Loken, et al., Clin. Nucl. Med. Vol. 10, No. 12, pp. 902-911, 1985). In The Loken Article, .sup.111 In-labeled leukocytes were used to assess occult infections in more than 1700 patients. Of the patients determined to have an occult infection, the sensitivity, specificity, and accuracy of the .sup.111 In-labeled leukocyte was determined to be 88%, 96%, and 94%, respectively.
.sup.111 In-labeled lymphocytes have been used to study the disease process in patients with chronic lymphocytic leukemia and well differentiated lymphoma. (Dutcher, Sem. Nucl. Med., Vol. 14, No. 3, 1984). In the Dutcher article, .sup.111 In-labeled lymphocytes were used to study the migration of carcinoma cells, normal lymphoid cells, and malignant lymphoid cells in patients with malignancy. Determining the migration of these cell types was beneficial in helping to understand the disease processes and the mechanism of metastasis.
.sup.111 In-labeled platelets were used for the evaluation of intracoronary thrombolysis and the quantitative estimation of platelet thrombosis on vascular grafts. (Dewanjee, Sem. Nucl. Med. Vol. 14, No. 3, 1984). In the Dewanjee article, .sup.111 In-labeled platelets were used to determine the number of adherent platelets on the deendothelialized surfaces of damaged cell walls and synthetic vascular grafts. Platelet deposition was recorded in denuded tissues, atherosclerotic vessels, and prostheses placed in the circulatory system. .sup.111 In-labeled platelets were also used to determine platelet consumption during open heart surgery. The article additionally described the in vivo evaluation of myocardial infarction using .sup.111 In-labeled leukocytes.
As indicated above, leukocytes and platelets can be successfully labeled with .sup.111 In. The current state of technology for labeling leukocytes with .sup.111 In involves the use of an .sup.111 In-indium oxine complex. The indium oxine portion of the .sup.111 In-indium oxine complex penetrates the cell membrane and carries the .sup.111 In into the cell interior.
One disadvantage of the .sup.111 In-indium oxine labeling method is that it is toxic to leukocytes. Studies have demonstrated decreased chemotaxis and increased leukocyte adherence after .sup.111 In-indium oxine labeling. (Shechan et. al., Inter J. Nucl. Med. Bio. Vol. 12 243-247, 1985; Linhart-Colas et. al., Brit. J. Hem. Vol. 53, pp. 31-34, 1981).
Another disadvantage of the .sup.111 In-indium oxine labeling method is that it is toxic to platelets. Decreased platelet aggregation has been noted after .sup.111 In-indium oxine labeling.
Yet another disadvantage of the .sup.111 In-labeling method is that .sup.111 In is extremely expensive. .sup.111 In is expensive because it is produced by a cyclotron. Therefore, clinical studies using .sup.111 In are often cost-prohibitive.
.sup.99m Tc, on the other hand, is an ideal imaging agent for labeling leukocytes and platelets. .sup.99m Tc is the most commonly used radioisotope in nuclear medicine. .sup.99m Tc is inexpensive ($0.35/mCi) because it is produced by a reactor.
Attempts to label leukocytes and platelets with .sup.99m Tc have failed. These attempts have resulted in elution (leaking) of the .sup.99m Tc from the cells. (Thakur, et al., Sem. in Nuc. med. Vol 14(2), 107-117, 1984). A review article published in 1984 noted that "the challenge of developing a .sup.99m Tc cell-labeling agent comparable to the .sup.111 In lipophillic chelates has still no: been met." (McAfee et al., Sem. in Nucl. Med. Vol. 14(2), p.82-105, 1984). Over the last few years many .sup.99m Tc agents have been proposed as leukocytes labels but none have enjoyed lasting success. (Peters, Nucl. Med. Comm. 8, 313-316, 1987).
Electroporation (electropermeation) involves the exposure of cells to a pulsed electric field. This electric field causes a dielectric breakdown of the cell membrane forming pores in the cell membrane. These pores allow the transfer of molecules from outside the cell into the cell interior. These pores seal upon the cessation of the electric field. Cells remain viable, and electron micrographic studies show no damage from the electroporation procedure. (Zimmerman, Rev. Physiol. Biochem. pharmacol., Vol. 105, pp. 175-257, 1986).
Electroporation is a technology with many different applications. One application of electroporation is the transfection of DNA into plant and mammalian cells. This technique was first performed in 1982, and since that time there has been much research in this area. (Chu, et. al., Nucl. Acid Res. Vol. 15(3), pp. 1311-1326, 1987; Eid, et. al., Proc. Natl. Acad. Sci. USA Vol. 84, pp. 7812-7816, 1987). In the Eid article, using electroporation, DNA having a molecular weight of four million was transfected into procaryotic cells. In another study using electroporation, molecules having molecular weights of between 9,000 and 154,000 passed through cell membranes. (Liang et al., Biotech. Vol. 6, No. 6, pp. 550-558, 1988). The molecules used by Liang et al. were fluorescently labeled dextrans. The label was used to monitor the extent of incorporation.
Erythrocytes have been labeled with C-14 sucrose by electroporation. (Kinosita et al., Nature vol. 27 , pp.258-260, 1978). These labeled erythrocytes were injected into a mouse where they remained in circulation with a normal average half life. There was no apparent elution of the C-14 sucrose from the erythrocytes.
Electroporated cells have been suggested as a new drug delivery system. (Zimmerman et al., Biochimica et. Biophysica. Acta, Vol. 436, pp. 460-474, 1976). In the Zimmerman article, the authors disclose a technique for loading an enzyme, urease, into human red blood cell ghosts (hemoglobin-depleted erythrocytes). In another article, molecules as large as tetrasacharides were loaded into viable erythrocytes (not hemoglobin-depleted ghosts) without affecting cell viability. (Tsong, et al., Biblthca. Haemat., No. 51, pp. 108-114, 1985).